1. Neuroscience

Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses

  1. Patricia M Horvath
  2. Michelle K Piazza
  3. Lisa M Monteggia  Is a corresponding author
  4. Ege T Kavalali  Is a corresponding author
  1. Department of Pharmacology, Vanderbilt University, United States
  2. Department of Neuroscience, the University of Texas Southwestern Medical Center, United States
  3. Vanderbilt Brain Institute, Vanderbilt University, United States
  4. Neuroscience Program, Vanderbilt University, United States
https://doi.org/10.7554/eLife.52852
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  1. Patricia M Horvath
  2. Michelle K Piazza
  3. Lisa M Monteggia
  4. Ege T Kavalali
(2020)
Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses
eLife 9:e52852.
https://doi.org/10.7554/eLife.52852
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Abstract

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Introduction

Synaptic neuronal communication can be broadly classified into either evoked or spontaneous neurotransmission. Evoked neurotransmission is the canonical action-potential driven signaling that causes synchronous or asynchronous release of vesicles at multiple synapses (Südhof, 2013). Spontaneous neurotransmission occurs via action-potential independent release of single synaptic vesicles. At the molecular level, spontaneous neurotransmission has been shown to utilize partly different molecular machinery and act at distinct postsynaptic sites than evoked neurotransmission (Kavalali, 2015).

The organizing principles surrounding evoked and spontaneous neurotransmission may differ between excitatory and inhibitory synapses. Spontaneous and evoked glutamate release at excitatory synapses in the hippocampus, as well as synapses at the Drosophila neuromuscular junction, activate distinct sets of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Melom et al., 2013; Peled et al., 2014; Sara et al., 2011). Synaptic N-methyl-D-aspartate (NMDA) receptors in the hippocampus also show a near complete segregation in their responses to spontaneous and evoked glutamate release (Atasoy et al., 2008; Reese and Kavalali, 2016). These studies have demonstrated that spontaneous and evoked neurotransmission can occur at the same synapse; nevertheless, these different forms of transmission activate separate NMDA and AMPA receptors. Segregation may be necessary due to the clear and distinct differences in downstream signaling between evoked and spontaneous neurotransmission at excitatory synapses (Autry et al., 2011; Crawford et al., 2017; Fong et al., 2015; Gonzalez-Islas et al., 2018; Nosyreva et al., 2013; Ramirez et al., 2017; Sutton et al., 2006; Sutton et al., 2007; Sutton et al., 2004). It remains unclear whether spontaneous and evoked neurotransmission play separate roles at inhibitory synapses. In contrast to excitatory neurotransmission, inhibitory neurotransmission serves distinct functions in circuits, targets different neuronal sites, and partly relies on separate presynaptic and postsynaptic molecular machineries (Courtney et al., 2018; Higley, 2014; Tyagarajan and Fritschy, 2014; Williams and Smith, 2018). Therefore, it is critical to address directly whether receptors that respond to spontaneous and evoked GABA release are segregated at inhibitory synapses.

Here, we examine whether inhibitory synapses exhibit postsynaptic segregation of spontaneous and evoked neurotransmission. These experiments require a use-dependent γ-aminobutyric acid-A receptor (GABAAR) antagonist to separate inhibitory spontaneous and evoked neurotransmission. Picrotoxin (PTX) is a commonly used noncompetitive GABAAR antagonist (Akaike et al., 1985; Gallagher et al., 1978; Nicoll and Wojtowicz, 1980; Takeuchi and Takeuchi, 1969). Structural evidence suggests PTX binds within the pore of the GABAAR (Chen et al., 2006; Gurley et al., 1995; Hibbs and Gouaux, 2011; Masiulis et al., 2019; Olsen, 2006), and may act in a use-dependent manner to block GABAAR channels (Akaike et al., 1985; Newland and Cull-Candy, 1992; Yoon et al., 1993). However, previous studies used exogenously applied GABA to examine the pharmacology of PTX; this setting may not be completely relevant to physiological signaling mediated by synaptically released GABA. We first show PTX acts as a use-dependent GABAAR antagonist during inhibitory neurotransmission. We subsequently used PTX to investigate postsynaptic segregation of spontaneous and evoked signaling at inhibitory synapses and identified partially segregated populations of GABAARs that are solely activated by evoked release. Collectively, these results provide new insight into fundamental aspects of GABAergic neurotransmission.

Results

Picrotoxin acts as a use-dependent antagonist in a manner consistent with open channel block

We first tested whether PTX acts as a use-dependent GABAAR antagonist to selectively block open GABAARs activated during inhibitory neurotransmission in dissociated hippocampal cultures. Typically, blockers that gain access to their binding sites via open channel pores prematurely hinder channel conductance leading to faster decay times. Therefore, in these experiments, we examined decay times after PTX treatment. Both spontaneous and evoked currents had faster decay times after PTX (Figure 1A–D). Here and all subsequent experiments, we used 50 µM of PTX as this concentration of PTX was able to completely abolish spontaneous miniature inhibitory postsynaptic currents (mIPSCs) within 5 min of application (Figure 1—figure supplement 1, also see below). Under the same conditions, stimulating hippocampal synapses at a variety of frequencies and measuring the evoked IPSC (eIPSC) peak amplitudes in the presence of PTX resulted in a response that decreased as a function of stimulation number regardless of PTX incubation time, indicating PTX is a use-dependent antagonist (Figure 1E). In contrast, stimulation in the absence of PTX led to a small decrease in eIPSC peak amplitude which was far less than the decrease in the presence of PTX, and may reflect metabolic rundown. Together, these data support PTX as use-dependent antagonist during inhibitory neurotransmission.

Figure 1 with 1 supplement see all
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PTX blocks GABAARs in a use-dependent manner consistent with open-channel block.

(A) Scaled example traces of mIPSCs before and after PTX addition. (B) Quantification showing average event decay times obtained from the same cell before and after (8 min) treatment with 50 µM PTX. mIPSC decay time is decreased following PTX treatment (paired t-test t(11) = 9.055, p<0.0001, n = 12). (C) Scaled example traces of evoked responses to 0.1 Hz stimulation before and after PTX addition. Average trace taken from the 10th response to stimulation in PTX following 8 min of PTX application at rest (no stimulation). (D) Quantification showing average evoked response decay time obtained from the same cell before and after (8 min) treatment with 50 µM PTX. Evoked response decay time is decreased following PTX treatment (paired t-test t(12) = 6.097, p<0.0001, n = 13). (E) (Left) PTX block of evoked response plotted by total treatment time. (Right) PTX block of evoked response plotted by stimulation number. PTX blocks evoked response as a function of stimulation number, rather than time, indicating it is a use-dependent blocker (non-linear regression single exponential fit for conditions with PTX; Time: Sum-of-Squares F test F(6, 141)=38.16, p<0.0001 that is one curve does not fit all datasets; Stimulation number: Sum-of-Squares F test F(6, 141)=1.005, p=0.4243 that is one curve does fit all datasets, n = 5 all groups). Decay of the eIPSC response without PTX is significantly less than with PTX, indicating that rundown is not responsible for the decrease in response. (non-linear regression single exponential fit for all conditions; Time: Sum-of-Squares F test F(12, 705)=115.9, p<0.0001 that is one curve does not fit all datasets; Stimulation number: Sum-of-Squares F test F(12, 705)=101.9, p<0.0001 that is one curve does not fit all datasets, 0.1 Hz - no PTX n = 46, 0.2 Hz - no PTX n = 11) Graphs are mean ± SEM. **** indicates p<0.0001.

Figure 1—source data 1

Source data for Figure 1.

https://cdn.elifesciences.org/articles/52852/elife-52852-fig1-data1-v2.xlsx

Kinetics of picrotoxin block correlate with presynaptic release probability at inhibitory synapses

If picrotoxin is truly a use-dependent antagonist, then the rate of GABAAR block should be proportional to presynaptic release probability (as with MK801 and NMDA receptors; Hessler et al., 1993; Rosenmund et al., 1993). To test this premise, we manipulated presynaptic release probability by altering external concentrations of Ca2+ in our dissociated culture system. Increasing Ca2+ concentration (from 0.5 mM – 8 mM) led to an increase in the initial eIPSC response to evoked stimulation (Figure 2A–B). Moreover, increasing Ca2+ concentration led to a decrease in paired pulse ratio and switched synapses from facilitation to depression, which is consistent with an increase in release probability (Figure 2A,C). We then stimulated neurons in the presence of PTX and recorded the progression of the eIPSC responses over 100 stimulations. Increasing release probability, via increasing external Ca2+ concentration, reliably led to a more rapid block of the eIPSC response (Figure 2D). These data demonstrate that PTX acts as a use-dependent blocker and can be used to monitor alterations in presynaptic release probability at inhibitory synapses.

Figure 2
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PTX can be used to compare release probability of inhibitory synapses.

(A) Example traces of paired pulse responses at an interstimulus interval of 100 ms in 0.5 mM Ca2+ (pulse one black, pulse two gray), 1 mM Ca2+ (pulse one pink, pulse two light pink), 2 mM Ca2+ (pulse one blue, pulse two light blue) or 8 mM Ca2+ (pulse one purple, pulse two light purple). (B) Quantification of initial peak amplitude of eIPSC in different Ca2+ concentrations. Increasing Ca2+ concentration increases the initial peak eIPSC amplitude, consistent with increased release probability (one-way ANOVA F(3,32) = 27.24, p<0.0001, Tukey’s post-hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.0160, 1 mM Ca2+ vs 2 mM Ca2+ p=0.0079, 2 mM Ca2+ vs 8 mM Ca2+ p=0.3821, 0.5 mM Ca2+ vs 8 mM Ca2+ p<0.0001, n = 9 all groups). (C) Paired pulse ratio recorded from cells in different Ca2+ concentrations. Increasing Ca2+ concentration decreased paired pulse ratio, consistent with increased release probability (two-way ANOVA interaction F(6,26) = 2.801, p=0.0308, interevent interval factor F(2,26) = 2.220, p=0.1287, Ca2+ concentration factor F(3,26) = 43.55, p<0.0001, Tukey’s post-hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.1202, 1 mM Ca2+ vs 2 mM Ca2+ p=0.0024, 2 mM Ca2+ vs 8 mM Ca2+ p=0.0107, 0.5 mM Ca2+ vs 8 mM Ca2+ p<0.0001, n = 3 for 0.5 mM Ca2+; n = 3 for 1 mM Ca2+; n = 3 for 2 mM Ca2+; n = 4 for 8 mM Ca2+). (D) eIPSC peak amplitude over successive 0.2 Hz stimulations in the presence of PTX. Increasing Ca2+ concentration increased the rate of eIPSC block. (Inset) Individual time constants of single exponentials fitted to each experiment. Increasing Ca2+ concentration decreased the time constant, consistent with an increased rate of block, demonstrating the utility of PTX to estimate release probability (one-way ANOVA F(3,38) = 5.125, p=0.0045, Tukey’s post-hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.4468, 0.5 mM Ca2+ vs 2 mM Ca2+ p=0.0162, 0.5 mM Ca2+ vs 8 mM Ca2+ p=0.0062, 2 mM Ca2+ vs 8 mM Ca2+ p=0.8203, n=10 for 0.5 mM Ca2+; n = 8 for 1 mM Ca2+; n = 14 for 2 mM Ca2+; n = 9 for 8 mM Ca2+). Graphs are mean ± SEM. * indicates p<0.05, ** indicates p<0.01, **** indicates p<0.0001, ns indicates not significant.

Figure 2—source data 1

Source data for Figure 2.

https://cdn.elifesciences.org/articles/52852/elife-52852-fig2-data1-v2.xlsx

GABAARs activated by spontaneous and evoked signaling show partial overlap

Our results indicate that PTX will only block GABAARs which have been activated. Taking advantage of the use-dependency of PTX, we designed a series of experiments in dissociated hippocampal cultures to evaluate the postsynaptic segregation of inhibitory evoked and spontaneous neurotransmission. Initially, we monitored a baseline of responses to evoked stimulation, then blocked all receptors activated by spontaneous neurotransmission with PTX incubation at rest and measured the remaining evoked response (Figure 3A). When measuring the time course of mIPSC block in PTX, we found that 5 min of PTX treatment in the absence of stimulation was sufficient to fully block the mIPSCs (Figure 3B–C). Both the frequency and amplitude distributions of these spontaneous events were unaltered by the addition of tetrodotoxin (TTX), indicating that suppression of excitatory synaptic transmission to isolate inhibitory neurotransmission is in itself sufficient to block all network activity and enable detection of mIPSCs without a requirement for TTX application (Figure 3—figure supplement 1). It is possible that some receptors which are activated by spontaneous release remain unblocked after 5 min, but these mIPSCs may be undetectable due to a reduced size. To test this possibility, we used a high Cl- internal solution to increase mIPSC amplitudes (Figure 3D–E). Augmented mIPSCs recorded using the high Cl- internal solution followed a similar time course of block in the presence of PTX as those recorded using the standard internal solution (Figure 3B–C), indicating that GABAARs which are activated by spontaneous signaling have reached a steady state of block following a 5 min incubation in PTX. Because PTX is use-dependent, and nearly all spontaneous responses are suppressed after a 5 min incubation (Figure 3B), the majority of remaining response to evoked stimulation after 5 min must be mediated by GABAARs which are only responsive to evoked neurotransmission.

Figure 3 with 1 supplement see all
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Evoked and spontaneous neurotransmission are partly segregated at inhibitory synapses.

(A) Schematic showing experiment design. (B) Time course indicating mIPSC block following the addition of PTX measured using standard internal solution (black) or high Cl- internal solution (gray). Integrated charge is binned in 20 s intervals. PTX diminished mIPSC frequency within 5 min. This time course is unchanged when measured using a high Cl- internal solution. (C) Example traces of mIPSC recordings from indicated time points in B. (D) Cumulative histogram of spontaneous event amplitudes in standard and high Cl- internal solutions. High Cl- internal solution shifted the distribution of mIPSCs toward higher amplitudes (Kolmogrov-Smirnov test D = 0.3350, p<0.0001, n = 1200 events from 12 standard internal solution recordings and 600 events from six high Cl- internal solution recordings, 100 events randomly selected per recording). (E) Average of spontaneous event amplitudes in standard and high Cl- internal solutions. High Cl- internal solution increased the average amplitude of mIPSC events (unpaired t-test t(1798) = 10.96, p<0.0001, n = 1200 events from 12 standard internal solution recordings and 600 events from six high Cl- internal solution recordings, 100 events randomly selected per recording). (F) Evoked inhibitory response to stimulation before drug treatment and following: no drug (open symbols, n = 6 for 2 min, n = 7 for 8 min), 1–8 min PTX (n = 5 for 1 min, n = 5 for 2 min, n = 5 for 5 min, n = 11 for 8 min), 8 min bicuculline (n = 4), or 8 min TTX treatment (n = 3). Treatment of the 8 min no drug condition with bicuculline after the 10th stimulation drastically reduced the response amplitude down to the level of 8 min bicuculline treatment, indicating that the measured response is mediated through GABAARs. Treatment with PTX for increasing amounts of time decreased the initial evoked response to stimulation, which continued to decay upon successive stimulations in all cases. However, initial evoked response was not further decreased after a 5 min treatment with PTX. (G) Example traces of initial evoked response after PTX treatment or rest (black = 2 min no PTX, gray = 8 min no PTX, green = 1 min PTX, pink = 2 min PTX, red = 5 min PTX, blue = 8 min PTX). (H) Quantification of the percent of the initial evoked response that is mediated by GABAARs which are unblocked following PTX treatment. Values are adjusted for bicuculline baseline and no drug treatment maximum response. After 5 min in PTX, when all receptors activated by mIPSCs are blocked, the unblocked evoked response is 40.1 ± 9.6% of the maximum response. This response is not further decreased following an 8 min treatment with PTX (39.7 ± 4.6%; one-way ANOVA F(3,22) = 6.228, p=0.0032, Tukey’s post-hoc testing 1 min vs 2 minutes p=0.2260, 1 min vs 5 minutes p=0.0124, 1 min vs 8 minutes p=0.0028, 5 min vs 8 minutes p>0.9999, n = 5 for 1 min, n = 5 for 2 min, n = 5 for 5 min, n = 11 for 8 min). Graphs are mean ± SEM. * indicates p<0.05, ** indicates p<0.01, **** indicates p<0.0001, ns indicates not significant.

Figure 3—source data 1

Source data for Figure 3.

https://cdn.elifesciences.org/articles/52852/elife-52852-fig3-data1-v2.xlsx

To evaluate whether suppression of mIPSCs also hinders subsequent evoked responses, we first established the upper and lower limits of evoked GABAergic responses to stimulation in our system. When no drug is applied during rest, the evoked response is diminished compared to the initial response before treatment (Figure 3F, open symbols). This may be due to metabolic rundown, as in these recordings we did not detect any alterations in membrane or pipette access resistances. Spontaneous mIPSCs, in contrast, were largely unaffected by this rundown (Figure 3—figure supplement 1). These data establish the upper bound of the GABAAR-mediated response. To establish a lower bound for GABAAR-mediated response, bicuculline was applied for 8 min. As a competitive antagonist, bicuculline blocks GABAARs regardless of whether they have been activated (Akaike et al., 1985; Masiulis et al., 2019). Response amplitudes after bicuculline incubation were greatly diminished (Figure 3F, purple), however, some current remained (~7%), which was sensitive to tetrodotoxin application, indicating that it was not an artifact of stimulation (Figure 3F, brown). This is consistent with previous studies, in which PTX achieved an imperfect block of GABA-induced current (Akaike et al., 1985; Newland and Cull-Candy, 1992; Yoon et al., 1993). Additionally, application of bicuculline to control drug-free conditions led to a drastic and immediate decrease in eIPSC amplitudes, indicating that responses above this baseline level represent currents mediated by GABAARs.

Next, we applied PTX in the absence of stimulation for 5 min, and then stimulated cells and measured the eIPSC response. If there is complete overlap between the receptors which are activated by spontaneous release and those activated by evoked release, after a 5 min incubation with PTX, when the majority of receptors activated by spontaneous release are blocked, we would expect to see no eIPSC response to stimulation above the level reached after bicuculline block. However, if the two populations of receptors are completely separate, we would expect to see a high eIPSC response comparable to the drug-free condition response. In these experiments, we found an intermediate initial eIPSC response following complete block of receptors activated by spontaneous release (Figures 3F–H, 5 minutes). The response decreased over successive stimulations due to the continued presence of PTX, indicating that receptors which were activated by previous evoked release are subsequently blocked. Using the initial responses in the drug-free condition and those remaining after bicuculline treatment as the maximum and minimum of the detectable GABAAR-mediated response, we were able to calculate that 40.1 ± 9.6% of the evoked response remains after complete suppression of mIPSCs. The magnitude of the remaining evoked response was remarkably similar if the cells were incubated at rest with PTX for 8 min (39.7 ± 4.6%, Figure 3H). This result indicates that approximately 40% of the evoked inhibitory response is mediated by postsynaptic GABAARs which are exclusively activated by evoked neurotransmission, while the remaining 60% of the response is mediated by receptors which are activated by both spontaneous and evoked neurotransmission in hippocampal cultures. Consistent with a partial overlap of receptors activated by evoked and spontaneous neurotransmission, the initial response to evoked stimulation was much higher when receptors activated by spontaneous release were not fully blocked (Figures 3F–H and 1–2 minutes).

Use dependence of PTX block of GABAergic transmission in hippocampal slices

To probe the postsynaptic organization of spontaneous and evoked neurotransmission within an intact synaptic circuit, we utilized ex vivo hippocampal slices from mature rats (11–13 weeks). We confirmed PTX’s use-dependency in hippocampal slice by measuring evoked field Inhibitory Postsynaptic Potentials (fIPSPs) within the CA1 region in response to varying concentrations of external Ca2+ (Figure 4A–E). Increasing extracellular Ca2+ concentration between 0.5 mM, 1 mM, and 2 mM caused an increase in release probability, as indicated by a decrease in paired pulse ratio (Figure 4B–C) and increase in initial peak amplitude of fIPSPs (Figure 4B,D). In the presence of PTX to block GABAARs, greater release probability caused an increase in the rate of GABAAR block as evidenced by a faster and more pronounced decline in peak amplitude of fIPSPs (Figure 4E). These data support our earlier conclusions by demonstrating that PTX is use-dependent and can be used to compare release probability across different conditions in hippocampal slice recordings.

Figure 4
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PTX exhibits use-dependency in hippocampal slices and demonstrates partial segregation of evoked and spontaneous neurotransmission at inhibitory synapses.

(A) Schematic showing experimental design in B-E. (B) Averaged fIPSP paired pulse representative traces at an interstimulus interval of 100 ms in 0.5 mM Ca2+ (pulse one black, pulse two gray), 1 mM Ca2+ (pulse one pink, pulse two light pink) or 2 mM Ca2+ (pulse one blue, pulse two light blue). (C) Paired pulse ratio (PPR) (P2/P1) was lower in 2 mM extracellular Ca2+ than in 0.5 mM Ca2+ or 1 mM Ca2+ and lower in 1 mM Ca2+ than 0.5 mM Ca2+, indicating that extracellular Ca2+ concentration is positively associated with presynaptic release probability (repeated measures two-way ANOVA F(2,48) = 45.96, p<0.0001, Tukey’s post hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.0145, 0.5 mM Ca2+ vs 2 mM Ca2+ p<0.0001, 1 mM Ca2+ vs 2 mM Ca2+ p<0.0001, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (D) Quantification of baseline peak amplitudes confirming that greater extracellular Ca2+ concentration increases presynaptic release probability and is associated with greater peak amplitude of fIPSPs (one-way ANOVA F(2,14) = 13.85, p=0.0005, Tukey’s post-hoc testing 0.5 mM Ca2+ vs 1 mM Ca2+ p=0.0072, 0.5 mM Ca2+ vs 2 mM Ca2+ p=0.0005, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (E) Time course showing block of 0.1 Hz evoked fIPSPs following PTX application in 0.5 mM Ca2+ (black), 1 mM Ca2+ (pink) or 2 mM Ca2+ (blue). Greater presynaptic release probability via increased extracellular Ca2+ is associated with faster block of GABAARs (non-linear regression single exponential fit, Sum-of-Squares F test F(6, 2048)=571.3, p<0.0001, n = 6 for 0.5 mM Ca2+, n = 5 for 1 mM Ca2+, n = 6 for 2 mM Ca2+). (F) Schematic showing experimental design in G-I. (G) Time course showing evoked fIPSP response to 0.1 Hz stimulation before (black) and following application of PTX at rest for 0 min (gray), 5 min (red) or 10 min (blue). Treatment of PTX for increasing amounts of time resulted in a lesser remaining response upon continuation of 0.1 Hz stimulation (n = 6 for 0 min, n = 7 for 5 min, n = 8 for 10 min). (H) Averaged fIPSP representative traces at baseline (black) and first fIPSP response following 0 min (gray), 5 min (red) or 10 min (blue) of PTX administration at rest. (I) Quantification of unblocked GABAAR mediating response remaining after 0, 5 or 10 min perfusion of PTX at rest, adjusted for upper and lower boundaries of fIPSP response. Following PTX administration at rest, the unblocked evoked response is 103.12 ± 3.82% of baseline after 0 min (gray), 88.0 ± 4.89% of baseline after 5 min (red) and 34.05 ± 5.37% of baseline after 10 min (blue) (one-way ANOVA F(2,18) = 56.30, p<0.0001, Tukey’s post-hoc testing 0 min vs 10 min p<0.0001, 5 min vs 10 min p<0.0001, n = 6 for 0 min, n = 7 for 5 min, n = 8 for 10 min). Thus a response to evoked stimulation remains following 10 min of GABAAR block by spontaneous neurotransmission, indicating partial segregation. Graphs are mean ± SEM. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001.

Figure 4—source data 1

Source data for Figure 4.

https://cdn.elifesciences.org/articles/52852/elife-52852-fig4-data1-v2.xlsx

Next, we utilized hippocampal fIPSP recording to examine postsynaptic cross talk of GABAARs activated by spontaneous and evoked release in hippocampal slices. Baseline responses were recorded for 10 min at 0.1 Hz stimulation followed by bath application of PTX at rest for 0, 5 or 10 min. We then continued to perfuse PTX and resumed 0.1 Hz stimulation for 30–40 min. (Figure 4F). Our previous data show that 5 min of PTX application at rest is sufficient to block spontaneous activity in cultured hippocampal neurons, and it has been reported that the rates of spontaneous neurotransmission are similar between cultured neurons and brain slices (Ertunc et al., 2007; Kavalali, 2015; Sara et al., 2005). Therefore, we measured the remaining evoked response in 5 min intervals following PTX application at rest, as the time for PTX to perfuse into the recording chamber and reach full concentration may differ between our culture and slice recordings. Accordingly, multiple minutes passed before the fIPSP response was reduced following the start of PTX perfusion when stimulation was continuously given (Figure 4E,G). Additionally, fIPSP peak amplitude was reduced compared to baseline when stimulation was resumed at 5 and 10 min, but the extent of block was much higher following 10 min of application at rest (Figure 4G–I). The decreased response following PTX application in the absence of stimulation indicates a high degree of overlap between GABAARs activated by evoked and spontaneous neurotransmission. Conversely, following 10 min of PTX application at rest, when most receptors activated by spontaneous signaling are expected to be suppressed, there is a remaining evoked response, suggesting that this response is mediated by GABAARs activated specifically by evoked stimulation consistent with our prior findings. Remarkably, this remaining response was similar in magnitude to our results in culture (34.05 ± 5.37%).

Recovery of spontaneous neurotransmission following GABAAR block

Our findings so far suggest that evoked and spontaneous neurotransmission partially overlap and that a population of GABAARs are solely activated by evoked release by examining the remaining evoked responses following GABAAR block during rest. To investigate this further, we examined the kinetics of recovery from GABAAR block in our dissociated culture system. For this purpose, we blocked GABAARs by applying PTX in the presence of 55 mM of KCl for 90 s then measuring the recovery of mIPSCs from PTX block in the presence or absence of stimulation (Figure 5A). As expected, the eIPSC response was greatly diminished when 55 mM of KCl alone was applied for 90 s, likely due to rapid depletion of vesicle pools (Sara et al., 2002). Nevertheless, the eIPSC responses recovered back to a steady baseline following removal of the KCl solution. eIPSC responses following 55 mM of KCl with PTX treatment also recovered following successive stimulations, however the recovery occurred over a longer time period, consistent with the premise that blocked receptors were gradually unblocked by successive stimulations (Figure 5B–C). This finding agrees with previous studies (Newland and Cull-Candy, 1992) where PTX unblock from GABAARs has been shown to be at least partially use-dependent (Figure 5—figure supplement 1). We also examined mIPSC responses to 55 mM of KCl with and without PTX in the presence or absence of this stimulation. Following application of 55 mM of KCl, mIPSC responses were partly diminished, but then recovered back to a steady baseline (Figure 5D–E). When 55 mM of KCl was co-applied with PTX, mIPSC responses were greatly diminished, indicating GABAARs activated by spontaneous release were blocked by 55 mM of KCl and PTX co-application. The recovery of mIPSCs in cells treated with 55 mM of KCl and PTX in the absence of stimulation was slow and minimal (Figure 5D–E). This is not surprising since the spontaneous release rate per synapse is in the order of 1 vesicle per minute per synapse (Leitz and Kavalali, 2014; Sara et al., 2005). However, the presence of 0.2 Hz stimulation significantly increased the recovery of mIPSCs (Figure 5F), indicating that evoked release activates some of the same GABAARs as spontaneous release. To ensure our results were not being biased towards receptors which are specifically activated by evoked release caused by depolarization, we repeated this experiment using the GABAAR agonist muscimol (10 µM) in combination with PTX to block GABAARs (Figure 6). We obtained similar results using this approach, that is stimulation significantly increased the recovery of mIPSCs following GABAAR block (Figure 6D–E). These results support the notion that GABAARs which are activated by spontaneous and evoked release partially overlap, but do not exclude the existence of a fraction of GABAARs which are only activated by spontaneous release.

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Recovery from PTX block of receptors activated by spontaneous GABA release is enhanced by the administration of evoked stimulation.

(A) Schematic showing experiment design. (B) Example traces of eIPSC responses from time points indicated in C. Stimulus artifacts were removed for clarity. (C) Quantification of eIPSC recovery following KCl treatment with and without PTX. KCl treatment initially depresses the eIPSC response. The evoked response is recovered after KCl treatment without PTX, but recovery after KCl treatment with PTX proceeds over a longer time course indicating GABAARs which were activated by KCl treatment and blocked by PTX are being unblocked with successive stimulations. (D) Example traces of mIPSCs from the timepoints indicated in E recorded in the presence or absence of stimulation before and after KCl treatment with and without PTX. (E) Quantification of mIPSC recovery (1 min bins) in the presence or absence of stimulation following KCl treatment with and without PTX. KCl treatment without PTX initially depresses mIPSCs, but does not fully block them. KCl treatment with PTX blocks mIPSCs, after which they recover slowly (KCl w/out PTX – no stim, n = 6; KCl w/out PTX – 0.2 Hz stim, n = 5; KCl w/PTX – no stim, n = 6; KCl w/PTX – 0.2 Hz stim, n = 6). (F) Analysis of mIPSC recovery (1 min bins) in PTX treated samples in the absence (open triangles) and presence (closed circles) of 0.2 Hz stimulation. Data were fitted with non-linear regression exponential model (no stimulation, dashed curve, n = 6; 0.2 Hz stimulation, solid curve, n = 6) which were significantly different from each other (Sum-of-squares F test F(2,109) = 6.976, p=0.0014). These data indicate that stimulation increases the recovery of mIPSCs, consistent with partial overlap of receptors activated by evoked and spontaneous signaling at inhibitory synapses. Graphs are mean ± SEM. ** indicates p<0.01.

Figure 5—source data 1

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Figure 6
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A similar pattern of mIPSC recovery from block is detected when using muscimol to activate GABAARs during PTX block.

(A) Schematic showing experiment design. (B) Example traces of eIPSC responses from time points indicated in C. Stimulus artifacts removed for clarity. (C) Quantification of eIPSC recovery following muscimol treatment with and without PTX (n = 4, all groups). Muscimol treatment initially depresses the eIPSC response. The evoked response is recovered after muscimol treatment without PTX, but recovery after muscimol treatment with PTX proceeds over a longer time course indicating GABAARs which were activated by muscimol treatment and blocked by PTX are being unblocked with successive stimulations. (D) Example traces of mIPSCs from the timepoints indicated in E. (E) Analysis of mIPSC recovery (1 min bins) in PTX treated samples in the absence (open triangles) and presence (closed circles) of 0.2 Hz stimulation. Data were fitted with non-linear regression exponential models (no stimulation, dashed curve, n = 6; 0.2 Hz stimulation, solid curve, n = 3) which were significantly different from each other (Sum-of-squares F test F(2,74) = 4.832, p=0.0107). These data indicate that stimulation increased the recovery of mIPSCs, consistent with partial overlap of receptors activated by evoked and spontaneous signaling at inhibitory synapses. (F) Graphic summary of findings indicating a partial overlap of receptors activated by spontaneous and evoked GABA release. Partial overlap could be achieved either through spatial segregation of evoked and spontaneous presynaptic release and postsynaptic receptors within the same synapse, or through specialization of synapses for either spontaneous release, evoked release, or both. Graphs are mean ± SEM. * indicates p<0.05.

Figure 6—source data 1

Source data for Figure 6.

https://cdn.elifesciences.org/articles/52852/elife-52852-fig6-data1-v2.xlsx

Discussion

In this study, we show that PTX blocks synaptic GABAARs in a use-dependent manner. This use-dependent action of PTX allowed us to investigate fundamental properties of GABAergic inhibitory synapses. By altering presynaptic release probability via changes in external Ca2+, we show that PTX can be used to interrogate release probability at inhibitory synapses in both cultures and hippocampal slices. Additionally, we were able to test whether evoked and spontaneous neurotransmission processes are segregated at inhibitory synapses. Our data show that while it is not as complete as previously reported at excitatory synapses (Atasoy et al., 2008; Melom et al., 2013; Peled et al., 2014; Reese and Kavalali, 2016; Sara et al., 2011), there is nevertheless significant postsynaptic segregation of the two forms of neurotransmission at inhibitory synapses.

The use-dependent NMDA receptor blocker MK-801 has been instrumental in determining basic properties of glutamatergic synapses such as presynaptic release probability (Hessler et al., 1993; Huang and Stevens, 1997; Huettner and Bean, 1988; Rosenmund et al., 1993), receptor saturation (McAllister and Stevens, 2000), and receptor mobility (Tovar and Westbrook, 2002). Our results show that PTX is similarly use-dependent and can be used to monitor alterations in release probability of GABAergic synapses. By increasing extracellular Ca2+ levels, we gradually augmented release probability at inhibitory synapses and observed a corresponding increase in the rate at which GABAergic responses were blocked. It is therefore likely that many of the same analyses initially performed for NMDA receptors at glutamatergic synapses can be performed for GABAARs at GABAergic synapses. However, it is important to caution that PTX does not simply block pore conductance of GABA channels but rather binds to a site within the pore to stabilize a closed state (Masiulis et al., 2019). In addition, recovery from block is only partially use-dependent as indicated by earlier work (Newland and Cull-Candy, 1992). Although these properties of PTX have limited impact on the experiments we performed here, they may influence alternative experimental designs such as delineating metabotropic and ionic receptor effects (Nabavi et al., 2013), or those that solely rely on response recovery after block.

Our results show that evoked and spontaneous neurotransmission processes are partially segregated at inhibitory synapses. This notion is supported by the GABAAR-mediated evoked responses remaining following the block of all GABAARs activated by spontaneous neurotransmission in both hippocampal cultures and slice. We estimate that 34–40% of the total evoked GABAergic response recorded at the stratum pyramidale of hippocampal CA1 region and in hippocampal cultures is mediated by receptors which are only activated by evoked neurotransmission. Although mIPSC recovery following GABAAR block was enhanced by the presence of stimulation, these results do not exclude the possibility that some GABAARs are only activated by spontaneous release and account for the population that recovers in the absence of stimulation. These findings suggest a model of partial, but not complete, segregation between GABAARs which are activated by evoked release, and those activated by spontaneous release (Figure 6F).

A major question arising from this work is: how is this partial segregation achieved? Segregation may start at the level of presynaptic release mechanisms, including dedicated signaling pathways that selectively impact spontaneous or evoked release as well as distinct synaptic vesicle pools and vesicle recycling pathways to carry out the two forms of neurotransmission (Abrahamsson et al., 2017; Kavalali, 2015). A growing body of evidence suggests that synapses are organized into ‘nano-columns’ such that a single vesicle only activates juxtaposed receptors on the postsynaptic membrane (Biederer et al., 2017; Maschi and Klyachko, 2017; Tang et al., 2016). Based on these studies, two non-mutually exclusive models can be proposed. One model posits that segregation is achieved through a combination of differential presynaptic molecular regulation and pre/postsynaptic spatial separation within the same synapse. Alternatively, segregation may arise through differential action of these mechanisms over different inhibitory synapses. Earlier studies demonstrated that excitatory synapses can exhibit both spontaneous and evoked release while others exclusively exhibit spontaneous or evoked release (Leitz and Kavalali, 2014; Melom et al., 2013; Peled et al., 2014; Reese and Kavalali, 2016; Walter et al., 2014). With GABAergic neurotransmission, limitations in the resolution and speed of current chloride sensors or other probes to visualize activity at the level of single synapses, makes this question difficult to address at this time. Therefore, we cannot exclude the possibility that some GABAergic synapses could release neurotransmitter only via spontaneous release, only via evoked release, or both leading to the partial segregation seen in our experiments (Figure 6F).

There could be many reasons why segregation is not as complete at inhibitory synapses as at excitatory synapses. First, there may be more overlap in the molecular machinery used for spontaneous and evoked neurotransmission at inhibitory synapses. Although earlier work has suggested that synaptic vesicle pools mediating spontaneous and evoked release may be distinct at inhibitory synapses (Chung et al., 2010), unlike at excitatory synapses, spontaneous release at inhibitory synapses is partly coupled to activation of voltage activated Ca2+ channels (Tsintsadze et al., 2017; Vyleta and Smith, 2011; Williams and Smith, 2018). Moreover, recent evidence suggests that synaptotagmin-1, the canonical Ca2+ sensor for fast synchronous evoked release (Geppert et al., 1994), also regulates Ca2+ dependent spontaneous release, but only at inhibitory synapses (Courtney et al., 2018). Second, unlike Ca2+ signaling through NMDA receptors, chloride flow through GABAARs has very few targets that may direct downstream biochemical signaling (Chen et al., 2019; Heubl et al., 2017; Piala et al., 2014), although GABAergic transmission has been shown to indirectly regulate dendritic Ca2+ signaling (Chiu et al., 2013; Higley, 2014). Therefore, the strong segregation that is seen at excitatory synapses may not be as essential to mediate inhibitory synaptic communication. Our current findings provide evidence that the postsynaptic organization of inhibitory synapses shows only partial segregation of GABAARs that respond to evoked and spontaneous neurotransmission (Figure 6F), suggesting a distinct organizing principle of these synapses compared to their excitatory counterparts. Nevertheless, given recently identified targets for chloride-mediated biochemical signaling in neurons (Chen et al., 2019; Heubl et al., 2017; Piala et al., 2014), even this partial segregation may provide a platform for differential signaling by evoked and spontaneous GABAergic neurotransmission in the central nervous system.

Materials and methods

Animals

Adult female dam CD1 rats (Sprague-Dawley, Charles River) aged 11–13 weeks were housed on a 12 hr light/dark cycle at ambient temperature (23 ± 3°C and humidity (50 ± 20%) with access to food and water ad libitum. Dams were used for hippocampal slice experiments. Rat pups (postnatal day (PN) 1–3) were used for culture experiments. Animal procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees at UT Southwestern Medical Center and at Vanderbilt University.

Cell culture

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Dissociated hippocampal cultures were prepared as previously described (Kavalali et al., 1999). Briefly, whole hippocampi were dissected from PN1-3 rats, trypsinized (~4 mg/mL, Sigma), mechanically dissociated, and plated on matrigel (BD Biosciences) coated glass coverslips. Neurons were plated in MEM containing 27.8 mM of Glucose, 2.4 mM of NaHCO3, 1.3 µM of Transferrin (Calbiochem), 2 mM of L-Glutamine, 4.4 µM of insulin, and 10% FBS. On days in vitro (DIV) 1, FBS concentration was reduced to 5%, L-Glutamine concentration was reduced to 500 µM, and 1x B-27 supplement (Gibco) and 4 µM of cytosine arabinoside (ARAC; Sigma) were added. On DIV4 the concentration of ARAC was reduced to 2 µM. Cells were maintained at 37°C in 5% CO2 atmosphere.

Hippocampal culture whole cell recordings

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Electrophysiology was performed as previously described (Nelson et al., 2006). On DIV15-21 cells were recorded using the whole cell voltage clamp configuration of the patch clamp technique. Extracellular Tyrode solution, adjusted to pH 7.4 and 319 mOsm, contained: 150 mM of NaCl, 4 mM of KCl, 1.25 mM of MgCl2·6H2O, 10 mM of glucose, 10 mM of HEPES, 2 mM of Ca2+ (unless otherwise noted), 50 µM of D-APV, and 10 µM of NBQX. Intracellular pipette solution, adjusted to pH 7.3 and 304 mOsm, contained: 115 mM of Cs-methanesulphonate, 10 mM of CsCl, 5 mM of NaCl, 10 mM of HEPES, 20 mM of TEA.Cl hydrate, 4 mM of MgATP, 0.3 mM of GTP, 0.6 mM of EGTA, and 10 mM of QX314. High Chloride intracellular pipette solution, adjusted to pH 7.3 and 304 mOsm, contained: 122 mM of CsCl, 9 mM of NaCl, 1.8 mM of MgCl2, 9 mM of EGTA, 9 mM of HEPES, 14 mM of creatine phosphate (Tris salt), 4 mM of MgATP, and 0.3 mM of Tris-GTP. Drugs in all experiments were used at the following concentrations unless otherwise noted: 50 µM of picrotoxin (Sigma Aldrich), 20 µM of bicuculline (Sigma Aldrich), 50 µM of D-APV (Abcam), 10 µM of NBQX (Abcam), 1 µM of TTX (ENZO Life Sciences), and 10 µM of muscimol (Tocris). Cells were held at −70 mV and liquid junction potential was not corrected for. eIPSCs were elicited by bath stimulation through parallel platinum electrodes. eIPSCs were on the order of 2–4 nA. Series resistance compensation was not employed in these experiments and series resistance measures were 10 MΩ or below. Series resistance was monitored before the start and following the conclusion of every recording. In cases where multiple files were recorded from the same patch, series resistance was monitored in between each file. For mIPSC data in Figure 1 and Figure 1—figure supplement 1, ‘before PTX treatment’ data were extracted from a 2 min spontaneous baseline recording before drug treatment, and ‘with PTX treatment’ data were extracted from the final 2 min of the 8 min drug treatment from the experiment in Figure 3F. For mIPSC data in Figure 3—figure supplement 1, ‘before TTX’ data were extracted from a 30 s spontaneous baseline recording before TTX treatment, and ‘after TTX’ was extracted from the final 30 s of a subsequent 10 min recording. Evoked inhibitory currents were analyzed using pCLAMP10 software (Molecular Devices). Spontaneous inhibitory currents were analyzed using MiniAnalysis software (Synaptosoft). For mIPSC time courses, integrated charge was measured as the summed area of each event within each bin.

Hippocampal slice electrophysiology

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Adult female dam rats aged 11–13 weeks were used for all hippocampal slice experiments. No more than four hippocampal slices were used per dam, and each experiment was performed on slices from at least two different animals. Dams were anesthetized with isofluorane and decapitated. Brains were removed and immersed in ice-cold dissection buffer containing the following: 2.6 mM of KCl, 1.25 mM of NaH2PO4, 26 mM of NaHCO3, 0.5 mM of CaCl2, 5 mM of MgCl2, 212 mM of sucrose and 10 mM of glucose for 2–3 min. Hippocampi were dissected and cut with a vibratome into 400 μm-thick transverse sections in ice-cold dissection buffer continuously aerated with 95% O2 and 5% CO2. Area CA3 was surgically removed immediately after sectioning. Sections were recovered for 3–6 hr at 30°C in oxygenated artificial cerebrospinal fluid (ACSF) containing the following: 124 mM of NaCl, 5 mM of KCl, 1.25 mM of NaH2PO4, 26 mM of NaHCO3, 2 mM of CaCl2, 1 mM of MgCl2 and 10 mM of glucose, pH 7.4 (continuously aerated with 95% O2 and 5% CO2). 25 μM of D-APV and 10 μM of NBQX were added to the ACSF to block NMDA and AMPA channels, respectively, and isolate inhibitory GABA-mediated currents. In several experiments, PTX was applied for long durations to fully block GABAARs. Following the conclusion of these experiments, bicuculline was applied to confirm the range of inhibitory response. Bicuculline did not further decrease the measured response, indicating that as for dissociated culture experiments, PTX blocked the full range of the GABAAR-mediated response. For experiments examining release probability, concentration of CaCl2 in ACSF varied at 2 mM, 1 mM and 0.5 mM. Following recovery, hippocampal slices were transferred to the recording chamber and perfused with oxygenated ACSF at a rate of 2–3 mL/min at 30°C. Field Inhibitory Postsynaptic Potentials (fIPSPs) were evoked by inserting a concentric bipolar stimulating electrode (FHC Inc) and an extracellular recording electrode filled with ACSF (resistance, 1–2 MΩ) proximally in s. pyramidale of CA1 just below the surface of the tissue Younts et al., 2016.

In all experiments, paired pulse ratio was measured by eliciting paired-pulse stimulations at decreasing interstimulus intervals (ISIs) of 500, 400, 300, 200, 100, 50, 20 and 30 ms. The fIPSP peak amplitude of pulse 2 (P2) was divided by pulse 1 (P1) to give a ratio representing presynaptic release probability. Baseline responses were then collected every 10 s (0.1 Hz) using an input stimulus intensity that induced 75% of the slice’s maximum response for 10 min. All data presented are the mean ± SEM of individual trials, and data are not binned. Specific n numbers for each experiment are specified in the figure legends.

To examine the use-dependency of picrotoxin (Figure 4A–E), extracellular Ca2+ in ACSF was varied between 0.5 mM, 1 mM and 2 mM to alter presynaptic release probability. 50 μM of picrotoxin was then perfused at a flow rate of 18.0 mL/hour with 0.1 Hz stimulation and fIPSP peak amplitude was recorded for 20 min to produce a decay curve of GABAAR block. 75% of maximal response varied from 85 to 223 μV response elicited by a 35–40 µA stimulation for 0.5 mM Ca2+, 231–297 μV response elicited by a 15–39 µA stimulation for 1.0 mM Ca2+, and 175–358 μV response elicited by a 12–15 µA stimulation for 2.0 mM Ca2+. To investigate evoked GABAAR activity following block of spontaneous GABAAR (Figure 4F–I), 50 μM of picrotoxin was perfused at a flow rate of 18.0 mL/hour at rest for 0, 5 or 10 min. 0.1 Hz stimulation was then resumed and picrotoxin perfusion continued for 40 min. 75% of maximal response varied from 175 to 358 µV response elicited by a 12–15 µA for 0 min, 208–361 µV response elicited by a 9–25 µA stimulation for 5 min, and 245–460 µV response elicited by a 11–18 µA stimulation for 10 min.

Data were analyzed by normalizing peak response amplitudes to average baseline peak amplitude for each recording. Unblocked receptor percentage following 0, 5 or 10 min PTX perfusion at rest was quantified by determining upper and lower limits of fIPSP response using the average of the first and last 50 stimulations of each recording, respectively. The lower limit was then subtracted from the first fIPSP response following the continuation of stimulation, and this value was divided by the range and subtracted from one to determine the percentage of baseline response that was intact. Slices were visually inspected before experiments and continually monitored throughout. Recordings with unstable initial baselines or presynaptic fiber volleys were excluded from analysis.

Statistical analyses

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All statistical analyses were performed using GraphPad Prism 7 or 8 software (GraphPad). Paired t-tests were performed to compare mIPSC frequency, mIPSC amplitude, mIPSC decay time, and evoked decay time before and after PTX addition. A non-linear regression was fitted to the block of eIPSCs and fIPSPs in PTX graphed by either time or stimulation number, and a sum-of-squares F test was used to determine if the curves were significantly different. One-way ANOVAs were used to compare initial eIPSC and fIPSP peak amplitude, time constants (τ) for different concentrations of external Ca2+, and percent not blocked evoked signal. Two-way ANOVAs were used to compare paired pulse ratio in different concentrations of external Ca2+. Tukey’s post-hoc testing was used where appropriate following ANOVAs. Unpaired t-tests were used to compare mIPSC frequency and amplitude with the high Cl- pipette solution and paired t-tests were used to compare before and after TTX application. A Kolmogrov-Smirnov test was used to compare frequency distributions for mIPSC amplitude with standard and high Cl- pipette solution. A linear regression was used to calculate the rate of eIPSC recovery following block with PTX in high KCl. A sum-of-squares F test was used to determine if the slopes of these lines were significantly different. A non-linear regression was used to calculate the rate of recovery of mIPSCs following block with PTX in high KCl and muscimol, and a sum-of-squares F test was used to determine if the curves were significantly different. Statistical significance was defined as p<0.05. Specifics of statistical tests employed are listed in figure legends.

Data availability

All source data files are included in the manuscript and supporting files.

References

    1. Gurley D
    2. Amin J
    3. Ross PC
    4. Weiss DS
    5. White G
    (1995)
    Point mutations in the M2 region of the Alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin
    Receptors & Channels 3:13–20.

Decision letter

  1. John Huguenard
    Reviewing Editor; Stanford University School of Medicine, United States
  2. Richard W Aldrich
    Senior Editor; The University of Texas at Austin, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The manuscript by Horvath et al. carefully documents a novel segregation of inhibitory synaptic receptors between those responsible for spontaneous (miniature) responses and those driving activity dependent release. This work builds on the power of dispersed ex vivo cultures of neurons that facilitates detailed physiological studies, and the exploitation of the fact that the channel blocking inhibitor picrotoxin can be used to provide information on recent use of receptors. Overall, these results show that similar to what is already known about excitatory synapses, inhibitory synapses show a similar subdivision into spontaneous vs evoked, although the segregation is less complete for inhibition.

Decision letter after peer review:

Thank you for sending your article entitled "Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses" for peer review at eLife. Your article is being evaluated by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation is being overseen by Richard Aldrich as the Senior Editor.

There is potential interest in this paper, however there are two major concerns. The full reviews are posted below. By the way, the identification of picrotoxin as a use-dependent blocker is a nice finding.

Can you briefly respond to the following:

1) The potential impact of this report might be limited given that the extent of segregation between spontaneous and evoked GABA-containing vesicles seems to be much less than for excitatory vesicles. This combined with some results that were perceived to be ambiguous in their interpretation potentially further weaken the paper.

2) The results obtained are limited to one preparation (immature monolayer culture) and it is unclear how general this finding is in relation to developmental stage and in the "more intact" nervous system.

Reviewer #1:

Horvath et al. , using the model system of immature rat hippocampal dispersed cultures, report on the potential independence of GABA-a receptors mediating spontaneous vs activity-dependent synaptic release of the inhibitory neurotransmittter GABA. They find that the GABA-a receptor pore blocking antagonist picrotoxin shows novel use-dependence that should enable the use of this compound to infer information about release probabilities at inhibitory synapses, given that high release probabilities will lead to relatively greater release of GABA and therefore greater use-dependent block.

Using this approach, the authors show that, as previously shown for excitatory synaptic receptors, inhibitory receptors show partial segregation i.e. that the receptors mediating spontaneous (activity-independent, presumably spontaneous single vesicle release events) do no overlap 100% with those activated by evoked synaptic transmission.

The results are novel, in that segregation of receptors has not, to my knowledge, been reported for inhibitory synapses, and could be important in terms of further understanding of the mechanisms regulating synaptic transmission in the brain. The experiments are carefully executed and illustrated, and are for the most part convincing. A minor limitation is that the results are limited to this elegant, yet artificial cell culture system that may not fully recapitulate the native perisynaptic environment in the brain. This last issue could reduce the potential impact of the report.

1) Validation in another neural system, such as acute or cultured brain slices would broaden the interpretation and impact.

Reviewer #2:

In this manuscript Horvath al., described experiments performed on cultured rat hippocampal neurons performed to investigate if there is postsynaptic segregation of evoked and spontaneous release at inhibitory synapses. The work is an extension of earlier studies from the labs of Drs Kavalali and Monteggia which have studied how evoked and spontaneous release differ at many synapses in the hippocampus. I do not believe that the manuscript is suitable for publication in its current form because of a number of uncertainties concerning experimental design and interpretation of the findings. My major concern relates to interpretation the experiments described in Figure 4 after which the authors propose that some GABA receptors are only activated by spontaneous release and unaffected by concurrent evoked release. The design of this experiment was set up to preferentially increase the block of receptors involved in evoked release. The 90 sec exposure to picrotoxin and had previously been shown (Figure 3B) to only block 50% of the minis. However, following the application of 90 mM potassium and picrotoxin there appeared to be almost complete block of both spontaneous and evoked IPSCs. After application, the recovery of spontaneous transmission appeared more impaired than recovery of evoked transmission. I believe this result indicates that the authors have shown that some GABA receptors are involved in only evoked release and that the GABA receptors involved in spontaneous release are also involved in it evoked release. In addition to addressing this question I have other specific questions for the authors:

1) Please describe, in the Materials and methods or an early part of the Results, how eIPSCs were elicited? Was it by field stimulation or some other method? The time of decay of eIPSC changed with PTX and time. When was the exemplar in 1C recorded (how long after application) and was that steady state?

2) Please provide data on eIPSC decrement with stimulation/frequency in the absence of PTX (see Figure 1E). This will facilitate our interpretation of data comparing synaptic activity at different time points in later experiments.

3) Is the Y axis in Figure 3A (and 4E) measurement of normalized mIPSC frequency or a product of frequency and amplitude? If the latter, please justify.

4) There are a number of occasions in which it would be interesting to know how mIPSCs changed over time since "metabolic rundown" that affected eIPSCs in Figure 3 could also affect mIPSCs. In fact, in Figure 3B there appears to be ~30% rundown in 2 mins of basal activity in this set of experiments. This is surprising since Figure 3—figure supplement 1 shows frequency and amplitude are unaffected by TTX in unmatched experiments. The case would be stronger if it was demonstrated within recordings that TTX did not affect frequency or amplitude. Perhaps the experiments used to make the brown circle data in Figure 3F can be analyzed to test this idea.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your revised article "Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses" for consideration by eLife. Your article has been reviewed by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Summary:

The manuscript by Horvath et al. nicely documents a novel segregation of inhibitory synaptic receptors between those responsible for spontaneous (miniature) responses and those driving activity dependent release. This work builds on the power of dispersed ex vivo cultures of neurons that facilitates detailed physiological studies, and the exploitation of the fact that the channel blocking inhibitor picrotoxin can be used to provide information on recent use of receptors. Overall, these results show that similar to what is already known about *excitatory* synapses, inhibitory synapses show a similar subdivision into spontaneous vs evoked, although the segregation is less complete for inhibition.

Overall, this is an important contribution to the literature, as it provides novel information of inhibitory signaling in the brain, which is critical in maintaining proper circuit balance. The study should be the starting point for further studies of mechanisms and consequences of such segregation.

The authors made the following significant changes to the manuscript:

1) They added field recordings from acute hippocampal slices and replicated key findings obtained from cell cultures.

2) They added new experiments using muscimol.

3) Investigated use-dependence of PTX unblock at 2 frequencies.

Revisions:

– The only remaining substantive critique is that the LFP approach for assessing inhibition is not a standard one, and more direct experiments utilizing intracellular experiments would certain be more compelling, although these are not strictly required.

– In addition, there are few, if any details regarding the precise methods of ensuring that the evoked LFP response accurately portrays inhibitory signaling. Missing details are the intensity of the stimulus, both in relative (% of maximal) and absolute (µA) values, number of trials averaged, confirmation of pharmacological dissection, rejection/inclusion criteria, etc.

– With the insertion of the new slice data in the manuscript, it is not clear whether the subsequent results were obtained in slices or dispersed culture.

– Whenever possible, include raw traces in every figure as examples.

– On Figure 5, the baseline of the evoked events should be visible.

– The muscimol experiments quite important, and they should be integrated into the main figures.

https://doi.org/10.7554/eLife.52852.sa1

Author response

There is potential interest in this paper, however there are two major concerns. The full reviews are posted below. By the way, the identification of picrotoxin as a use-dependent blocker is a nice finding.

Can you briefly respond to the following:

1) The potential impact of this report might be limited given that the extent of segregation between spontaneous and evoked GABA-containing vesicles seems to be much less than for excitatory vesicles. This combined with some results that were perceived to be ambiguous in their interpretation potentially further weaken the paper.

Our results demonstrate that segregation of postsynaptic GABA receptors responding to evoked and spontaneous GABA release is not as complete as our previous reports on NMDA receptors and AMPA receptors (Atasoy et al., 2008; Sara et al., 2011). We think elucidating this fundamental property of GABAergic neurotransmission, regardless of the experimental outcome is extremely important. In our opinion, it was surprising that we could detect any segregation at all as there is limited evidence for biochemical signaling mediated by GABAergic transmission. Our earlier studies have associated segregation of evoked and spontaneous excitatory transmission with their distinct effects on biochemical signaling. Our current findings suggest a partial dissociation of putative signaling functions for evoked and spontaneous GABAergic neurotransmission, which we plan to pursue in future work.

Moreover, we believe the approach and the methods we demonstrate here will enable other groups to test the same question in their systems. For instance, our earlier work was instrumental in studies that were performed in the Drosophila neuromuscular junction by other groups (e.g. Peled et al., 2014).

2) The results obtained are limited to one preparation (immature monolayer culture) and it is unclear how general this finding is in relation to developmental stage and in the "more intact" nervous system.

We thank the reviewer for this suggestion. Our extensive work using hippocampal culture preparations show that synapses in this system reach maturity within two weeks as judged by their functional as well as ultrastructural properties (Kavalali et al., 1999; Mozhayeva et al., 2002). A key benefit of the culture systems is the ability to stimulate a large majority of the synaptic contacts onto the neuron of interest. Nevertheless, we have tested key predictions of these results in acute hippocampal slices and found a similar portion of the inhibitory evoked response remains after incubation with picrotoxin at rest to block GABAARs activated by spontaneous release. These results indicate that across systems 34-40% of the inhibitory evoked response is mediated by GABAARs which are only activated by evoked release and strengthen a key finding of our study.

Reviewer #1:

Horvath et al. , using the model system of immature rat hippocampal dispersed cultures, report on the potential independence of GABA-a receptors mediating spontaneous vs activity-dependent synaptic release of the inhibitory neurotransmittter GABA. They find that the GABA-a receptor pore blocking antagonist picrotoxin shows novel use-dependence that should enable the use of this compound to infer information about release probabilities at inhibitory synapses, given that high release probabilities will lead to relatively greater release of GABA and therefore greater use-dependent block.

Using this approach, the authors show that, as previously shown for excitatory synaptic receptors, inhibitory receptors show partial segregation i.e. that the receptors mediating spontaneous (activity-independent, presumably spontaneous single vesicle release events) do no overlap 100% with those activated by evoked synaptic transmission.

The results are novel, in that segregation of receptors has not, to my knowledge, been reported for inhibitory synapses, and could be important in terms of further understanding of the mechanisms regulating synaptic transmission in the brain. The experiments are carefully executed and illustrated, and are for the most part convincing. A minor limitation is that the results are limited to this elegant, yet artificial cell culture system that may not fully recapitulate the native perisynaptic environment in the brain. This last issue could reduce the potential impact of the report.

1) Validation in another neural system, such as acute or cultured brain slices would broaden the interpretation and impact.

We thank the reviewer for their kind words regarding our work and thoughtful suggestions. We have conducted additional experiments to test key predictions in hippocampal slice. Using this system and measuring fIPSPs we were able to recapitulate our findings from culture. As in culture, PTX acted in a use-dependent manner and was able to distinguish between different presynaptic release probabilities based on the rate of fIPSP block. Additionally, following incubation with PTX at rest to block GABAARs activated by spontaneous signaling, we found that 34 ± 5.37% of evoked response remains. This is remarkably similar to our results in culture, in which 40.1 ± 9.6% of the evoked response remained following incubation with PTX at rest.

Reviewer #2:

In this manuscript Horvath al., described experiments performed on cultured rat hippocampal neurons performed to investigate if there is postsynaptic segregation of evoked and spontaneous release at inhibitory synapses. The work is an extension of earlier studies from the labs of Drs Kavalali and Monteggia which have studied how evoked and spontaneous release differ at many synapses in the hippocampus. I do not believe that the manuscript is suitable for publication in its current form because of a number of uncertainties concerning experimental design and interpretation of the findings. My major concern relates to interpretation the experiments described in Figure 4 after which the authors propose that some GABA receptors are only activated by spontaneous release and unaffected by concurrent evoked release. The design of this experiment was set up to preferentially increase the block of receptors involved in evoked release. The 90 sec exposure to picrotoxin and had previously been shown (Figure 3B) to only block 50% of the minis. However, following the application of 90 mM potassium and picrotoxin there appeared to be almost complete block of both spontaneous and evoked IPSCs. After application, the recovery of spontaneous transmission appeared more impaired than recovery of evoked transmission. I believe this result indicates that the authors have shown that some GABA receptors are involved in only evoked release and that the GABA receptors involved in spontaneous release are also involved in it evoked release.

We thank the reviewer for their insightful comments. In our experiments, 90 seconds of 55 mM of potassium was used to elicit a flood of GABA neurotransmitter that would activate GABAARs beyond those normally activated by evoked release. We believe the increased block of mIPSCs supports this premise. However, the reviewer makes a good point that 90 seconds of 55 mM of potassium may not be the best way to block all GABAARs, and may preferentially block those receptors which mediate responses to evoked release. Therefore, we have performed additional experiments utilizing the GABAAR agonist muscimol to activate and block GABAARs. Our experiments with muscimol recapitulate our findings with KCl, and indicate that this manipulation did not selectively block receptors activated by evoked release.

Our interpretation of the data in this figure relies on the recovery of the GABAergic response following PTX block being use-dependent (as shown by new data in the supplement and described above). If the receptors activated by evoked and spontaneous release are overlapping, we would expect stimulation to activate the same receptors as spontaneous release, thereby speeding up the recovery of the spontaneous mIPSC response. However, if the receptors activated by evoked and spontaneous release are non-overlapping, we would expect stimulation to activate a separate set of receptors and therefore have limited impact on the recovery of the spontaneous mIPSC response, which would instead rely only on spontaneous signaling.

Initially, using our non-standard measure of mIPSC recovery we did not find an impact of stimulation on the rate of mIPSC recovery and thus concluded this represented a set of receptors which was only activated by spontaneous signaling. Upon reanalysis of our data to use the more standard integrated charge as a measure of GABAergic recovery, we did find that stimulation increased the rate of mIPSC recovery. This is more in line with our previous block experiments showing partial overlap of the GABAARs activated by spontaneous and evoked release. However, this data does not exclude the possibility that the recovery of mIPSCs in the absence of stimulation is partly mediated by receptors which are only activated by spontaneous release. The slow recovery of mIPSCs in the absence of stimulation is likely due to the slow rate of spontaneous release at any given synapse (~1 vesicle/minute) compared to the 0.2 Hz applied stimulation (1 stimulation/5 seconds).

In addition to addressing this question I have other specific questions for the authors:

1) Please describe, in the Materials and methods or an early part of the Results, how eIPSCs were elicited? Was it by field stimulation or some other method? The time of decay of eIPSC changed with PTX and time. When was the exemplar in 1C recorded (how long after application) and was that steady state?

We thank the reviewer for their careful examination of our study. The eIPSCs were elicited by bath stimulation through parallel platinum electrodes. The exemplar in 1C was taken from the 10th response to stimulation in PTX following 8 minutes of PTX application at rest (no stimulation). We have clarified this point in the text.

2) Please provide data on eIPSC decrement with stimulation/frequency in the absence of PTX (see Figure 1E). This will facilitate our interpretation of data comparing synaptic activity at different time points in later experiments.

We thank the reviewer for this insightful point. We agree that this will be useful to plot these data together and have adjusted our figures accordingly. As seen in Figure 1E, the rundown associated with stimulation in the absence of PTX is far less than the block of GABAergic response measured in the presence of PTX.

3) Is the Y axis in Figure 3A (and 4E) measurement of normalized mIPSC frequency or a product of frequency and amplitude? If the latter, please justify.

The Y-axes in these figures is a normalized product of mIPSC frequency and amplitude. We initially chose this metric as a measure of charge transfer because the integrated charge of each event would be affected by decreased decay time following PTX addition. This would decrease overall charge transferred without necessarily providing a full measure of receptor block. However, we recognize that this is a non-standard measure and have updated our analysis to reflect integrated charge.

4) There are a number of occasions in which it would be interesting to know how mIPSCs changed over time since "metabolic rundown" that affected eIPSCs in Figure 3 could also affect mIPSCs. In fact, in Figure 3B there appears to be ~30% rundown in 2 mins of basal activity in this set of experiments. This is surprising since Figure 3—figure supplement 1 shows frequency and amplitude are unaffected by TTX in unmatched experiments. The case would be stronger if it was demonstrated within recordings that TTX did not affect frequency or amplitude. Perhaps the experiments used to make the brown circle data in Figure 3F can be analyzed to test this idea.

The reviewer makes an excellent point and we have included experiments in which spontaneous IPSCs were measured within recordings before and after TTX. These recordings show that the frequency and amplitude of events is unchanged following the addition of TTX indicating that we are measuring bona fide mIPSCs. Additionally, mIPSCs are stable in TTX over the course of 10 minutes indicating that they are not affected by the same rundown which effects evoked IPSCs.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Revisions:

– The only remaining substantive critique is that the LFP approach for assessing inhibition is not a standard one, and more direct experiments utilizing intracellular experiments would certain be more compelling, although these are not strictly required.

While this may not be a standard approach, it allowed us to test the prediction of our findings in mature hippocampal slices. We have followed as closely as possible guidelines from other published works measuring inhibition in hippocampal slice (Younts et al., 2016). The ability of picrotoxin to block this response further indicates that it is a measureable inhibitory response. Unfortunately, due to institutional restrictions, we are not able to perform additional experiments at this time. However, we appreciate the critique and in future work, we will definitely test the premise of this study using whole cell voltage clamp methods in distinct GABAergic synapses in the CNS.

– In addition, there are few, if any details regarding the precise methods of ensuring that the evoked LFP response accurately portrays inhibitory signaling. Missing details are the intensity of the stimulus, both in relative (% of maximal) and absolute (µA) values, number of trials averaged, confirmation of pharmacological dissection, rejection/inclusion criteria, etc.

We have added these details and others to our Materials and methods section on hippocampal slice electrophysiology.

– With the insertion of the new slice data in the manuscript, it is not clear whether the subsequent results were obtained in slices or dispersed culture.

We have updated the text to provide clarity as to whether experiments in each section were performed in culture or slices.

– Whenever possible, include raw traces in every figure as examples.

We have now included example traces in all main figures and most supplemental figures.

– On Figure 5, the baseline of the evoked events should be visible.

We have adjusted our example traces in this figure to include the baseline both before and after the evoked events. For clarity, the stimulus artifact was removed from these traces, as well as those in Figure 6.

– The muscimol experiments quite important, and they should be integrated into the main figures.

We have moved this data into a newly created figure, Figure 6, to better highlight these results.

https://doi.org/10.7554/eLife.52852.sa2

Article and author information

Author details

  1. Patricia M Horvath

    1. Department of Pharmacology, Vanderbilt University, Nashville, United States
    2. Department of Neuroscience, the University of Texas Southwestern Medical Center, Dallas, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Project administration
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9969-8637

  2. Michelle K Piazza

    1. Vanderbilt Brain Institute, Vanderbilt University, Nashville, United States
    2. Neuroscience Program, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7852-3456

  3. Lisa M Monteggia

    1. Department of Pharmacology, Vanderbilt University, Nashville, United States
    2. Vanderbilt Brain Institute, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration
    For correspondence
    lisa.monteggia@vanderbilt.edu
    Competing interests
    Reviewing editor, eLife
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0018-501X

  4. Ege T Kavalali

    1. Department of Pharmacology, Vanderbilt University, Nashville, United States
    2. Vanderbilt Brain Institute, Vanderbilt University, Nashville, United States
    Contribution
    Conceptualization, Supervision, Funding acquisition, Project administration
    For correspondence
    ege.kavalali@vanderbilt.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1777-227X

Funding

National Institute of General Medical Sciences (T32 GM008203)

  • Patricia M Horvath

National Institute of Mental Health (R01 MH070727)

  • Lisa M Monteggia

National Institute of Mental Health (R01 MH66198)

  • Ege T Kavalali

National Institute of Mental Health (T32 MH064913)

  • Michelle K Piazza

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors would like to thank Dr. Ryan Hibbs for discussions at initial stages of this project. We would also like to thank Drs. Natali Chanaday, Ji-Woon Kim, Kanzo Suzuki, Melissa Bawa and PeiYi Lin and Robert Altamirano for comments during the preparation of this manuscript. This work was supported by National Institute of Health grants GM008203 (PMH), MH064913 (MKP), MH081060 and MH070727 (LMM), and MH66198 (ETK).

Ethics

Animal experimentation: Animal procedures conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center ( Animal Protocol Number APN 2016-101416) and at Vanderbilt University School of Medicine (Animal Protocol Number M1800103).

Senior Editor

  1. Richard W Aldrich, The University of Texas at Austin, United States

Reviewing Editor

  1. John Huguenard, Stanford University School of Medicine, United States

Version history

  1. Received: October 18, 2019
  2. Accepted: May 13, 2020
  3. Accepted Manuscript published: May 13, 2020 (version 1)
  4. Version of Record published: May 26, 2020 (version 2)

Copyright

© 2020, Horvath et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Patricia M Horvath
  2. Michelle K Piazza
  3. Lisa M Monteggia
  4. Ege T Kavalali
(2020)
Spontaneous and evoked neurotransmission are partially segregated at inhibitory synapses
eLife 9:e52852.
https://doi.org/10.7554/eLife.52852

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